D-2S, D-2L, D-3, and D-4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 x mesencephalon hybrid cell (MES-23.5) via distinct G proteins

We utilized the approach of stably expressing different dopamine (DA) recep tors into identified cell lines in an attempt to better understand the coup ling of these receptors to membrane ion channels via second messenger syste ms. Recently, we examined the N18TG2 X mesencephalon (MES-23.5) cell line t hat is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-depend ent K+ current present in these cells. Untransfected MES-23.5 cells display ed a voltage-dependent slow-onset, slowly inactivating outward current whic h was not altered by bath application of either the D-2 DA receptor agonist quinpirole (QUIN; 10-100 mu M) or the D-1 DA receptor agonist SKF38393, in dicating that these cells were devoid of DA receptors. The K+ current studi ed was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected, to stably express the D-2S, D-2L, D-3, and D-4 receptors, the same current wa s observed. In cells expressing D2L, D-2S, and D-3 receptors, application o f the DA receptor agonists QUIN (1-80 mu M), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 mu M), and dopamine (DA, 1-80 mu M), increased the peak o utward current by 35-40%. In marked contrast, cells stably expressing the D -4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D-2-like recept or antagonist sulpiride (SUL 5 mu M), when coapplied with QUIN (10 mu M), t otally abolished the change in K+ current normally observed, while coapplic ation of the D-1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D-2L, D-3, and D-4 receptor stimulation was pre vented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml fo r 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes G(o alpha) completely blocked the ability of D-2L, D-3, and D-4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against G(o alpha) w as without effect, whereas intracellular application of an antibody recogni zing G(s alpha) abolished tile ability of the D-2S receptor to enhance K+ c urrent. These findings demonstrate that different members of the D-2 DA rec eptor family may couple in a given cell to a common effector in dramaticall y different ways. Synapse 31:108-118, 1999. (C) 1999 Wiley-Liss, Inc.